Extracellular Vesicles (EVs) from Fully “Charged” MSCs Treat Advanced Cellular Models of Liver Fibrosis

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Blog Summary of a Webinar Presentation by Dr. Stefania Bruno, U. Torino

Fibrotic disease processes combine into a major unmet medical need, hefting an oversized burden on people suffering from atherosclerosis, long COVD, chronic kidney disease (CKD), and nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH), and other conditions. In fact, 45% of all mortality is driven by severe fibroproliferative disease processes of the inner organs and circulatory system. 1, 2, 3 Sadly, agency-approved treatment options to directly antagonize fibrotic molecular pathways are still not widespread, resulting in a large waiting list of patients facing a deficit of transplant-ready organs. 4 Despite this, public interest in fibrosis-targeting treatments has remained flat relative to disease classes like oncology or inflammation. 5 Nevertheless, abundant promising preclinical data with mesenchymal stem/stromal cells (MSCs) or their secreted extracellular vesicles (MSC-EVs; sometimes called “exosomes”) support their advancement into human trials. Associate Professor of Laboratory Medicine Dr. Stefania Bruno of the University of Torino (unito.it) is at the leading edge of progress-minded investigators who are discovering new ways to get MSC-EVs ready for prime time in the broad struggle with fibrotic disease.

RoosterBio’s Development Awards aim to support worldwide investigators who share our common goals to advance hMSC and exosome-based therapeutics by directing up to $10,000 in RoosterBio products for innovative study proposals. Dr. Bruno was selected out of a large applicant pool for one such award in 2022 for a project titled, “Evaluation of the anti-fibrotic activity of extra-cellular vesicles derived from different stem cell sources.” We were thus thrilled and honored to learn that Professor Bruno would be generously able to present some preliminary findings in a recent webinar, broadcast on Thursday, April 18, 2024, titled “Anti-fibrotic Effects of Extracellular Vesicles Produced by MSCs Maintained in Chemically Defined Medium.”

The Basics

Although her lab has also been keenly interested in chronic kidney disease, Dr. Bruno began the seminar by updating viewers with basic background information on liver fibrosis, or scarring. 6 This pathology is driven by an imbalance in extracellular matrix synthesis and degradation. After different types of hepatic injury (ischemia, oxidative stress, toxins), the release of inflammatory cytokines will induce activation of hepatic stellate cells to differentiate into myofibroblast that secrete collagen. Activated hepatic stellate cells express a-smooth muscle actin (a-SMA) and secret extracellular matrix (ECM) components (e.g., collagen).7

MSCs’ secreted factors and vesicles are, alone, capable of facilitating liver regeneration, anti-inflammation, and anti-fibrosis. The MSC secretome posits a therapeutic advantage, because these harvested products could be more readily mass-produced, collected, QC’ed, dosed, and stockpiled than their parental cell of origin. Extracellular vesicles (EVs) are an important secretome component of MSCs 8, 9 and also stem cells. 7 These are small, virus-sized membrane-bound particles that are released by cells and thought to play a crucial role in cell-cell communication. They display protein ligands or enzymes that function as signal transduction mediators and also carry “cargo” material (like microRNA, mRNA, bioactive lipids) between cells.10, 11 EVs can be naturally found in various body fluids, including blood, milk, urine, and saliva. They help regulate many normal bodily processes and are also involved in the progression of some diseases. 12, 13 Many questions remain, however, pertaining to the correct dosing of extracellular vesicles, as well as their best cell sources and expansion culture conditions.

Two In Vitro Extracellular Vesicle Models to Test Drive Their Biofunction Against Simulated Liver Fibrosis

In her ongoing research, Dr. Bruno made use of a two-dimensional (2D) and a three-dimensional (3D) model of liver fibrosis. In the 2D model, a line of hepatic stellate cells (LX-2) was cultured and activated with TGF-β1, which is a known mediator of fibrosis. Upon activation, LX-2s increase the expression of fibrosis markers like collagen and alpha-smooth muscle actin (α-SMA).

While the 2D model is useful for screening purposes, it doesn’t optimally mimic the complex environment of the liver. To more closely recapitulate a disease state in vitro, Dr. Bruno’s team also devised a 3D model using liver spheroids. These spheroids are small, spherical clusters of liver cells composed of hepatic stellate cells and hepatocytes (modeled by HepG2 cells). Similar to the 2D model, the 3D spheroids were treated with TGF-β1 to induce molecular hallmarks of fibrosis. The aim was to determine whether EVs from different preparations (3 per tissue) and tissue origins (bone marrow or umbilical cord) of MSCs could ablate the fibrotic response, as measured by mRNA expression of a-SMA, Collagen I, or TGF-b. Different doses were employed, as well.

MSC-EVs Made to Order from RoosterBio Bone Marrow & Umbilical Cord MSCs & Specialized Media

Using RoosterBio’s xeno-free RoosterVial™ cells (3 x umbilical cord MSC donors and 3 x bone marrow MSC donors), expansion medium (RoosterNourish™), and extracellular vesicles collection medium (RoosterCollect™), Dr. Bruno and colleagues prepared EVs from six different cell sources. Media were conditioned with RoosterCollect™ for 48h before collection, clarification, and isolation by ultracentrifugation (Figure 1).

Figure 1

Figure 1. Scheme for preparation and quality analysis of extracellular vesicles (EVs) from mesenchymal stem/stromal cells via umbilical cord (UC) or bone marrow (BM) sources.

These preparations were thoroughly characterized by nanoparticle tracking analysis (NTA) for size distribution of the populations, transmission electron microscopy (TEM) for morphology, and flow cytometry (MacsPLEX® Exosome Kit, Miltenyi Biotec) for distinguishing surface markers of EVs and MSCs. Consistent with other preparations of MSC-EVs, all exhibited similar sizes, morphologies, and surface marker expression patterns showing expression of tetraspanins (CD9, CD63, CD81) and putative identity markers of MSCs. 14

In 2D & 3D, MSC-EVs Suppress Simulated Liver Fibrosis Models

Extracellular vesicle treatments reduce TGFb-stimulated a-SMA and Collagen a1 in 2D and Collagen a1 and TGFb autocrine looping in the 3D spheroids as measured by qPCR. Professor Bruno and her group at U. Torino are thus demonstrating that MSC-EVs could be reliably isolated and administered to suppress molecular hallmarks of fibrosis in two different cell culture systems. Extracellular vesicles from both umbilical cord as well as bone marrow seemed to have an effect. To see this data presented more fully, visit the webinar recording.

Future Research Directions

Professor Bruno concluded the recorded webinar by acknowledging the contributions of Dr. Giulia Chiabotto, PhD, Dr. Elena Ceccotti, PhD, and Dr. Armina Semnani, graduated PhD student.

Dr. Bruno’s lab’s future research will aim to focus on several key areas to overcome current barriers to the use of extracellular vesicles in clinical settings against fibrotic disease:

  1. Improving the 3D Model: Enhancing the 3D liver model by incorporating additional cell types, such as macrophages, to better mimic the inflammatory environment of the liver.
  2. Human Hepatocytes: Using primary human hepatocytes instead of cell lines to create a more accurate model of human liver function and fibrosis.
  3. Molecular Characterization: Conducting detailed analyses of the molecular content of EVs to identify specific microRNAs and proteins that contribute to their anti-fibrotic effects.
  4. Animal Studies: Testing the efficacy and safety of EVs in animal models of liver fibrosis to pave the way for future clinical trials.

This webinar closed with a well-attended, live Q&A session with 14 questions on the methods, data, and future directions of this valuable work. To carefully follow the question and answer session of this webinar, please visit RoosterBio’s webinar portal for this event.

RoosterBio is very grateful for the exciting opportunity to welcome Stefania Bruno to share her latest (and ongoing) data with us and our viewers. With that said, we cannot resist mentioning that RoosterBio’s 2024 Development Award applications are imminent! Please stay tuned on our website or follow us on LinkedIn to stay notified on how you can help you can advance your lab’s work.

 

References
  1. Wynn, T. A., Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007, 117 (3), 524-9. 10.1172/JCI31487
  2. Wynn, T. A., Cellular and molecular mechanisms of fibrosis. J Pathol 2008, 214 (2), 199-210. 10.1002/path.2277
  3. Thannickal, V. J.; Zhou, Y.;  Gaggar, A.; Duncan, S. R., Fibrosis: ultimate and proximate causes. J Clin Invest 2014, 124 (11), 4673-7. 10.1172/JCI74368
  4. Friedman, S. L.; Sheppard, D.;  Duffield, J. S.; Violette, S., Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med 2013, 5 (167), 167sr1. 10.1126/scitranslmed.3004700
  5. RoosterBio hMSCs & Their Exosome/EV Secretomes — Time to Think Globally with Locally Acting Anti-Fibrotic Therapies. https://www.roosterbio.com/blog/hmscs-their-exosome-ev-secretomes-time-to-think-globally-with-locally-acting-anti-fibrotic-therapies/.
  6. Bruno, S.; Chiabotto, G.; Camussi, G., Extracellular Vesicles: A Therapeutic Option for Liver Fibrosis. Int J Mol Sci 2020, 21 (12). 10.3390/ijms21124255
  7. Chiabotto, G.; Ceccotti, E.;  Tapparo, M.;  Camussi, G.; Bruno, S., Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype. Front Cell Dev Biol 2021, 9, 777462. 10.3389/fcell.2021.777462
  8. Grignano, M. A.; Bruno, S.;  Viglio, S.;  Avanzini, M. A.;  Tapparo, M.;  Ramus, M.;  Croce, S.;  Valsecchi, C.;  Pattonieri, E. F.;  Ceccarelli, G.;  Manzoni, F.;  Asti, A.;  Libetta, C.;  Sepe, V.;  Iadarola, P.;  Gregorini, M.; Rampino, T., CD73-Adenosinergic Axis Mediates the Protective Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Ischemic Renal Damage in a Rat Model of Donation after Circulatory Death. Int J Mol Sci 2022, 23 (18). 10.3390/ijms231810681
  9. An, S.; Anwar, K.;  Ashraf, M.;  Lee, H.;  Jung, R.;  Koganti, R.;  Ghassemi, M.; Djalilian, A. R., Wound-Healing Effects of Mesenchymal Stromal Cell Secretome in the Cornea and the Role of Exosomes. Pharmaceutics 2023, 15 (5). 10.3390/pharmaceutics15051486
  10. Valadi, H.; Ekstrom, K.;  Bossios, A.;  Sjostrand, M.;  Lee, J. J.; Lotvall, J. O., Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007, 9 (6), 654-9. 10.1038/ncb1596
  11. Couch, Y.; Buzas, E. I.;  Di Vizio, D.;  Gho, Y. S.;  Harrison, P.;  Hill, A. F.;  Lotvall, J.;  Raposo, G.;  Stahl, P. D.;  Thery, C.;  Witwer, K. W.; Carter, D. R. F., A brief history of nearly EV-erything – The rise and rise of extracellular vesicles. J Extracell Vesicles 2021, 10 (14), e12144. 10.1002/jev2.12144
  12. Kalluri, R.; LeBleu, V. S., The biology, function, and biomedical applications of exosomes. Science 2020, 367 (6478). 10.1126/science.aau6977
  13. Wei, W.; Ao, Q.;  Wang, X.;  Cao, Y.;  Liu, Y.;  Zheng, S. G.; Tian, X., Mesenchymal Stem Cell-Derived Exosomes: A Promising Biological Tool in Nanomedicine. Front Pharmacol 2020, 11, 590470. 10.3389/fphar.2020.590470
  14. Witwer, K. W.; Van Balkom, B. W. M.;  Bruno, S.;  Choo, A.;  Dominici, M.;  Gimona, M.;  Hill, A. F.;  De Kleijn, D.;  Koh, M.;  Lai, R. C.;  Mitsialis, S. A.;  Ortiz, L. A.;  Rohde, E.;  Asada, T.;  Toh, W. S.;  Weiss, D. J.;  Zheng, L.;  Giebel, B.; Lim, S. K., Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications. J Extracell Vesicles 2019, 8 (1), 1609206. 10.1080/20013078.2019.1609206

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